Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

Role of accessory cells in B cell activation. IV. Ia+ accessory cells are required for the in vitro generation of thymic independent type 2 antibody responses to polysaccharide antigens

It has been previously shown that the in vitro antibody response to TNP-Ficoll requires the presence of adherent accessory cells. In order to determine if this characteristic was unique to TNP-Ficoll or a general feature of the TI-2 antibody responses, responses to the polysaccharide antigens TNP-Levan and TNP-Dextran were studied. Also, it was determined if the functionally relevant accessory cell expresses Ia determinants. Passage of spleen cells over Sephadex G-10 abrogated the response to TNP-Levan and TNP-Dextran as well as to TNP-Ficoll. Addition of adherent accessory cells to the G-10 passed spleen cells reconstituted the response to all 3 antigens. Pretreatment of the adherent accessory cells with a specific anti-Ia serum plus complement abrogated the ability of these cells to provide accessory cell function in the responses to all 3 antigens. Thus, an Ia-positive adherent accessory cell is required for the generation of TI-2 antibody responses to these polysaccharide antigens. This raises the possibility that genetic restrictions may exist between the Ia-positive accessory cell and the lymphocytes involved in the responses to TNP-Ficoll, TNP-Dextran, and TNP-Levan.     

Intraepithelial lymphocytes modulate Ia expression by intestinal epithelial cells

We have demonstrated that although intestinal epithelial cells in fetuses and young rats do not express Ia antigens, in adult rats intestinal epithelial cells do express Ia antigens, as indicated by immunoperoxidase staining with monoclonal antibodies. Ia expression by intestinal epithelial cells appeared to be related to an increase in the number of intraepithelial lymphocytes (IEL). Most of the IEL were T cells and expressed the phenotype associated with cytotoxic/suppressor T cells, and a large number contained cytoplasmic granules. To directly study a possible modulating effect of IEL on intestinal epithelium, an Ia-negative intestinal epithelial cell line (IEC 17) of rat origin was cultured in the presence of supernatants obtained from Con A- or PHA-stimulated lymphocytes. IEL, as well as spleen cells but not bone marrow cells, were able to secrete a factor(s) capable of inducing Ia antigens on IEC 17 cells, as judged by immunoperoxidase staining and radioimmunoassay. Ia-positive IEC 17 cells were detectable after 12 hr and maximum Ia expression was obtained by 48-hr incubation. Persistence of Ia expression by intestinal epithelial cells required the continued presence of Ia-inducing factor in the medium. Lymphocyte proliferation was not essential for the secretion of the Ia-inducing factor(s). The characteristics and the kinetics of secretion of the Ia-inducing factor were similar to that of an interferon-like activity, but not of interleukin 2. Con A-induced supernatants from IEL and spleen cells were also capable of suppressing the growth of IEC 17 cells. The results of this study indicate that IEL, because of their close association with intestinal epithelial cells, may be involved in modulating a variety of epithelial cell functions, including the expression of Ia antigens. This leads us to speculate that Ia-positive epithelial cells, like Ia-positive macrophages and dendritic cells, may be involved in antigen presentation to T lymphocytes.     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.     

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

IL-4 is required to generate and sustain in vivo IgE responses

Antibodies of the IgE isotype play a predominant role in immediate hypersensitivity reactions. IL-4, a T cell-derived lymphokine that stimulates increased Ia expression by resting B cells and increased IgG1 secretion by LPS-activated B cells in vitro, has also been shown to regulate in vitro and in vivo polyclonal IgE responses. We report that large quantities of a purified anti-IL-4 mAb inhibit primary in vivo polyclonal IgE responses by 99% in mice infected with Nippostrongylus brasiliensis or injected with anti-IgD antibodies, and totally inhibit secondary Ag-specific IgE responses to TNP-keyhole limpet hemocyanin without effect on either IgG1 or IgG2a responses to these stimuli. The lack of effect of anti-IL-4 antibody on IgG1 secretion cannot be explained simply by inadequate neutralization of IL-4, inasmuch as the doses of anti-IL-4 antibody used blocked an N. brasiliensis-induced increase in B cell Ia expression by more than 85%, whereas in vitro studies indicate that enhancement of B cell Ia expression requires less IL-4 than induction of IgG1 secretion. In addition to demonstrating that IL-4 plays a necessary role in the generation of an in vivo IgE response, we show that IL-4 has an important role in sustaining established IgE responses, because anti-IL-4 antibody, when given at the peak of an N. brasiliensis- or TNP-keyhole limpet hemocyanin-induced IgE response, accelerates the declines in total serum IgE and in IgE anti-TNP antibody levels, respectively. These observations suggest that the effects of IL-4 on in vivo immune responses may be more specific than might have been predicted from in vitro observations, and that regulation of IL-4 production or action may be useful for the prevention or therapy of immediate hypersensitivity disorders.     

Expression of I-A and I-E,C region-coded Ia antigens on functional B cell subpopulations.

Ia antigens from specific subregions have been examined on functional B cell populations. Expression of both I-A and I-E,C region antigens was demonstrated on cells required for both lipopolysaccharide mitogenesis and polyclonal activation. Similar I-A and I-E,C subregion expression was found on cells required for response to the T-independent antigen, polyvinylpyrrolidone. TNP-specific IgM and hen egg lysozyme-specific IgG plaque-forming cells also express I-A and I-E,C region antigens. No evidence was found for an Ia- population responsive in the systems tested. Further, no evidence of preferential expression of I-A or I-E,C region antigens was observed in any system examined. Therefore, it appears that B cells express both I-A and I-E,C region-coded Ia antigens.     

Large, activated B cells are the primary B-cell target of 8-bromoguanosine and 8-mercaptoguanosine

Previous studies have documented the ability of 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo) to induce polyclonal proliferation and differentiation of B cells from a variety of mouse strains. In the present study, we have defined the cellular target of this mitogenic activity. Using B cells fractionated according to size, we have found that large B cells are responsive to 8-BrGuo- and 8-MGuo-induced proliferation and differentiation whereas small, resting B cells are relatively unresponsive to these compounds. Addition of splenic adherent cells to the small B-cell fraction partially restored the proliferative but not the differentiative responses to 8-BrGuo and 8-MGuo. Although small B cells alone did not proliferate or differentiate in response to 8-BrGuo and 8-MGuo, cell surface expression of Ia antigens increased following incubation with these compounds. Thus, the biological activity of 8-BrGuo and 8-MGuo appears to be dictated by the cell type upon which it is acting. Small B cells are activated as evidenced by increased levels of surface Ia whereas large B cells are not only activated but are also induced to proliferate and differentiate.     

The syngeneic mixed leukocyte reaction: the genetic requirements for the recognition of self resemble the requirements for the recognition of antigen in association with self

We have used cells from inbred strain 2 and strain 13 guinea pigs in order to define further the role of Ia antigens in the syngeneic mixed leukocyte reaction (MLR). The guinea pig syngeneic MLR resembled the autologous MLR in man in that it demonstrated both memory and specificity. The Ia antigens appeared to be the proliferative stimuli in that the primary stimulator cell was an Ia-positive adherent peritoneal exudate cell (PEC) and the reaction could be specifically inhibited by anti-Ia sera directed to the stimulator cell. We also demonstrated the existence of two (2 x 13)F1 T cell populations that were capable of reacting to one or the other parental PEC in the absence of any known exogenous antigen. These results suggest that the syngeneic MLR may represent T cell activation mediated through a receptor for self Ia.     

Two roles for Ia in antigen-specific T lymphocyte activation

In this study we examined the mechanism by which a PPD-specific murine T cell hybridoma, 8B2, recognized PPD associated with antigen-presenting cells (APC) in a manner genetically restricted by I-Ad. It was found that PPD-pulsed APC that were glutaraldehyde-fixed and treated with anti-Ia monoclonal antibody (abbreviated as PGM) were unable to stimulate the 8B2 T cells, as expected, due to inhibition caused by antibody binding to the Ia. However, addition of non-antigen-treated, glutaraldehyde-fixed APC (abbreviated as G) to cultures containing 8B2 T cells and PGM restored T cell activation, as determined by IL 2 production. This second non-antigen-specific function provided by the additional APC, G, was attributed to Ia and could be substituted by APC plasma membranes and by soluble membrane extracts. Genetic restriction analysis in which a variety of Ia-positive and Ia-negative cell lines and B cell blasts from different mouse strains were used as PGM or as G showed that each APC provided different Ia determinants that were specifically recognized by the T cells. PGM cells had to express I-Ad in order to present the PPD determinant, whereas the non-antigen-specific function was specific for I-Ad or I-Ab. These results suggest that the anti-Ia antibody does not interfere with the PPD/I-Ad-specific determinant bound by the antigen-specific T cell receptor, but prevents a second non-antigen-specific interaction with another region of the Ia molecule, which is provided by G. These two roles for Ia (antigen-specific and non-antigen-specific) were also found for activation of normal polyclonal PPD-specific T cell responses; thus they are not unique to the 8B2 T cell, but are generally applicable. In addition, T cell interactions with PGM and with G each provide different intracellular activation signals. This was determined by substituting the PGM or the G with either the tumor promoter phorbol 12-myristate 13-acetate (PMA) or the Ca++ ionophore, ionomycin. It was found that 8B2 T cells cultured with PGM and ionomycin, but not with PGM and PMA, were activated for IL 2 production. Neither PMA nor ionomycin in conjunction with G resulted in T cell activation. Taken together, these results indicate that 8B2 T cell activation involves APC Ia antigens in two different ways: one is to contribute to the presentation of the foreign PPD antigen, and a second is a non-antigen-specific Ia-T cell interaction necessary to provide additional intracellular activation signals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigens on human monocytes identified by monoclonal antibodies

Two antigens (Mo1 and Mo2) present on human peripheral blood monocytes have been defined by lytic IgM monoclonal antibodies. Both antigens are present on greater than 70% of adherent mononuclear cells (predominantly monocytes). Mo1 is expressed by monocytes, granulocytes, and Null cells, but is absent from T and B lymphocytes. Mo2, on the other hand, appears specific for peripheral blood monocytes. Neither antigen is present on Ia-positive B cell lines or on tumor cells from patients with B cell lymphoproliferative malignancies, further excluding the possibility that Mo1 and Mo2 are Ia antigens. Mo1 and Mo2 are, however, present on a significant number of blast cells from patients with monocytic leukemia (both myelomonocytic and pure monocytic variants), but relatively infrequently expressed by cells from patients with acute granulocytic leukemia. These results indicate that Mo1 and Mo2 are unique antigens that may represent distinct stages of late monocyte-granulocyte differentiation.     

Polyclonal B cell activation by B cell differentiation factor B151-TRF2. I. Involvement of self-Ia recognition process mediated by B cells

The present study examined the functional role of Ia antigens on B cells in polyclonal B cell activation induced by a B cell differentiation factor, B151-TRF2. The polyclonal IgM PFC responses by B151-TRF2 were inhibited by monoclonal antibodies specific for class II MHC antigens (Ia antigens) but not class I MHC antigens. Such inhibition by anti-Ia antibodies was haplotype-specific and was observed in the absence of both T cells and accessory cells. Moreover, the anti-Ia antibody-induced inhibition of the B151-TRF2 responses was not due to the blocking of binding of B151-TRF2 to the corresponding B cell receptor. A series of kinetic studies revealed that some Ia-mediated cellular activation process occurs before the resting B cells become responsive to B151-TRF2. Thus, the B151-TRF2-mediated B cell responses consist of at least two distinct phases. The early phase is an Ia-dependent but B151-TRF2-independent process, whereas the late phase is an Ia-independent but B151-TRF2-dependent process. To further characterize the functional role of Ia antigens on B cells, an additional experiment was carried out by using F1 B cells which co-dominantly express both parental Ia antigens on the surface. Interestingly, it was observed that the degree of inhibition of the B151-TRF2-mediated responses of F1 B cells by anti-parental Ia antibody was, at best, one-half that of the parental B cells, suggesting that F1 B cells may be separated into two subpopulations with the restriction specificity for the respective parental Ia antigens. To examine this possibility, (B10 X B10.BR)F1 B cells were separated into adherent and nonadherent cell populations by their ability to bind to either one of the parental B cell monolayers, and the specificity of inhibition of their responses to B151-TRF2 by anti-Ia antibodies was assessed. It was found that the responses of (B10 X B10.BR)F1 B cells adherent to the B10 B cell monolayer or the B10.BR B cell monolayer were almost completely inhibited by anti-I-Ab and anti-I-Ak antibodies, whereas those of nonadherent cells were now selectively inhibited by anti-I-Ak and anti-I-Ab antibodies, respectively. These findings are interpreted as indicating that the B151-TRF2-responsive F1 B cells consist of at least two subpopulations with the restriction specificity for either one of the parental Ia antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nature of the antigenic complex recognized by T lymphocytes. IV. Inhibition of antigen-specific T cell proliferation by antibodies to stimulator macrophage Ia antigens.

We have previously demonstrated that nonimmune guinea pig T lymphocytes could be specifically sensitized with TNP-modified allogeneic macrophages after eliminating the alloreactive T cells with bromodeoxyuridine (BUdR) and light treatment. This procedure allowed the unique opportunity to use anti-Ia sera directed against the Ia antigens of only the stimulator macrophages or responder T cells to determine against which cell type anti-Ia would block TNP-specific stimulation. It was found that the TNP-specific DNA synthetic response of BUdR and light-treated T cells stimulated with TNP-modified allogeneic macrophages was totally eliminated by anti-Ia sera directed solely against the allogeneic stimulator macrophage. In contrast, anti-Ia sera directed only against the responder T cells had no effect on their response to TNP-modified allogeneic macrophages. These findings indicate that macrophage Ia antigens are required for efficient T cell-macrophage interactions and raise the possibility that T cell Ia antigens may not be required for collaboration with macrophages. This latter possibility was substantiated by experiments in which we show that treating T cells with anti-Ia sera and complement to remove the Ia-positive cells either before or after priming, or both, had no effect on their ability to be primed and restimulated with TNP-modified macrophages.     

Sodium periodate-induced T cell mitogenesis: an analysis of the requirement for Ia and IL 1

T lymphocytes oxidized with the mitogen sodium periodate undergo a proliferative response when cultured in the presence of Ia+ accessory cells. However, the exact role(s) the accessory cells play in such a response has not been clearly defined. We have evaluated the role of Ia and the requirement for interleukin 1 (IL 1) in periodate mitogenicity by using the Ia+ cloned tumor cell lines P388AD (Ia+, IL 1 inducible) and P388NA (Ia+, IL 1 noninducible) as accessory cells. P388AD but not P388NA was able to supply accessory cell function to periodate-treated T cells, suggesting that Ia expression alone was not sufficient to reconstitute a response. Monoclonal anti-I-Ad and anti-I-Ed antibody blocked the accessory cell function of P388AD. In addition, monoclonal antibody GK 1.5, directed against the T cell determinant L3T4a, blocked the P388AD/periodate-treated T cell interaction, confirming that this interaction was restricted by class II molecules. Although Ia expression was required, the response was not major histocompatibility complex (MHC) restricted, because allogeneic as well as syngeneic macrophages were capable of supplying accessory cell function to periodate-treated T cells. Exogenous IL 1 alone was able to trigger periodate-treated T cells, suggesting that Ia was required for the induction of IL 1 synthesis by the accessory cells. Furthermore, purified IL 2, devoid of IL 1 activity, was able to fully reconstitute the proliferative response of accessory cell-depleted oxidized T cells to a level equal to that of whole spleen accessory cells or P388AD. These data suggest that periodate-treated T cells can proliferate with IL 1 alone and that Ia+ accessory cells in periodate-mediated T cell mitogenicity may function in the release of IL 1 and the induction of IL 2 synthesis by the T cells.     

Influenza viruses as lymphocyte mitogens. II. Role of I-E molecules in B cell mitogenesis by influenza A viruses of the H2 and H6 subtypes

Influenza A viruses of the H2 and H6 subtypes behave as T cell-independent B cell mitogens for lymphocytes from strains of mice that express the class II MHC glycoprotein I-E (Ia.7+ haplotypes). We have examined the role of I-E molecules in mitogenesis by these viruses. Lymphocytes from (Ia.7+ X Ia.7-)F1 hybrid strains that express lower levels of I-E antigen than homozygous Ia.7+ strains showed a level of response to H2 and H6 influenza viruses that was intermediate between the high response of the Ia.7+ parent and the low response of the Ia.7- parent. The mitogenic response of H-2k lymphocytes to these viruses was completely inhibited by low concentrations of anti-I-Ek monoclonal antibody that had no effect on B cell proliferation induced by LPS or by influenza A virus of the H3 subtype. Furthermore, incubation of H-2k spleen cells with high concentrations of H2 (but not H3) influenza viruses substantially inhibited the binding of radio-labeled anti-I-Ek, but not anti-I-Ak, monoclonal antibody. Cell mixing experiments indicated that expression of I-E by the B cells was critical to the mitogenic response, whereas I-E expression by accessory cells may not be necessary. The data support a model in which B cell mitogenesis by these viruses results from direct binding of the viruses to I-E molecules on B lymphocytes.     

Patterns of expression of human "Ia-like" antigens during the terminal stages of B cell development.

The "Ia-like" antigens characteristically present on the membrane of B lymphocytes were shown to be absent in most terminally differentiated Ig-producing plasma cells. This was most evident in analyses of myeloma plasma cells that uniformly lacked the Ia-antigens. Also, in B lymphoid cell lines, the lymphoblasts were uniformly Ia-positive but the plasma cells were negative. In contrast, however, the majority of plasma cells produced after pokeweed mitogen stimulation remained positive. Plasma cells in tonsil and in the tissue of peripheral blood of patients with Wladenstr?m's macroglobulinemia were also sometimes Ia-positive although the majority were negative. Studies of membrane IgM and IgD indicated a similar loss in some instances. However, the SIg and Ia antigens were not invariably associated. These results lead to the conclusion that Ia-antigens are differentiation antigens for B cells and are usually lost by the end stage cells in this series.     

Role of antigen-specific T cell help in the generation of in vivo antibody responses. I. Antigen-specific T cell help is required to generate a polyclonal IgG1 response in anti-IgD antibody-injected mice.

A system in which injection of mice with an antibody to mouse IgD that they recognize as foreign stimulates a large, T cell-dependent IgG response was used to study whether Ag-specific T cell help is required to stimulate polyclonal (non-Ag-specific) IgG production in vivo. Igha x Ighb allotype heterozygous mice were injected with a conjugate of a foreign Ag coupled to a mAb specific for one of the two IgD allotypes expressed in these mice. This conjugate cross-links mIgD on B cells that express the recognized allotype. These cells process the conjugate and present the foreign Ag to Ag-specific T lymphocytes, which become activated. Thus, B cells of the recognized allotype can be stimulated by cross-linking of their mIgD, Ag-specific T cell help, non-Ag-specific cytokines, and non-Ag-specific contact with activated T cells. In contrast, B cells that express the Igh allotype not recognized by the Ag-anti-IgD antibody conjugate (bystander B cells) can be stimulated in this system only by non-Ag-specific cytokines and non-Ag-specific contact with activated T cells. Although both recognized and bystander B cells in conjugate-injected mice demonstrated substantial increases in size and Ia expression, only the recognized B cells were induced to synthesize DNA and to make a substantial polyclonal Ig response. Bystander B cells still failed to secrete IgG when mice were injected with an anti-IgD-Ag conjugate specific for the other Igh allotype as well as a mAb that cross-linked IgD of the bystander B cell allotype. These observations demonstrate that although non-Ag-specific cytokine and contact-mediated T cell help are sufficient to induce B cells to increase in size and Ia expression in anti-IgD antibody-injected mice, Ag-specific T cell help is required to stimulate the generation of an IgG response in these mice.     

Effect of interferon-gamma and human alpha 2-macroglobulin on peritoneal macrophage morphology and Ia antigen expression.

While the primary role of the plasma protein alpha 2-macroglobulin (alpha 2M) appears to be related to its proteinase inhibitory activity, alpha 2M has been reported to regulate the immune response in vitro. Previous studies have demonstrated that, although native alpha 2M has no effect on macrophage function, proteinase- or CH3NH2-treated alpha 2M antagonize the IFN-gamma-induced expression of class II major histocompatibility complex (Ia) antigens on mouse peritoneal macrophages. In this investigation, we examined the effects of alpha 2M-CH3NH2 on the IFN-gamma-induced expression of macrophage Ia antigens by indirect immunofluorescence microscopy, radioimmunoassay, and immunoprecipitation of biosynthetically-labelled Ia. While alpha 2M-CH3NH2 suppressed the IFN-gamma induced increase in the percentage of Ia-positive macrophages detected by immunofluorescence microscopy, alpha 2M-CH3NH2 had no effect on the average of number of Ia molecules expressed per cell as detected by radioimmunoassay. In addition, alpha 2M-CH3NH2 had no effect on the ability of IFN-gamma to induce biosynthesis of Ia. Microscopic examination of IFN-gamma-treated macrophages revealed that treatment with alpha 2M-CH3NH2 prevented IFN-gamma-induced changes in macrophage morphology. IFN-gamma-treatment of elongated inflammatory macrophages was associated with the generation of round cells which possessed few cytoplasmic projections. By contrast, addition of alpha 2M-CH3NH2 to the incubation prevented the IFN-gamma-induced morphological changes, and the cells remained elongated with irregular cytoplasmic borders. We postulate that alpha 2M-CH3NH2 decreases the IFN-gamma-induced expression of Ia by preventing morphological changes in macrophages, resulting in the distribution of existing Ia over a larger surface area. As a consequence of this, the perceived fluorescence intensity of the bound antibody is lowered and the cells appear to be Ia-negative.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)